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Forum: Sample Prep / Library Generation 01-28-2020, 07:36 AM
Replies: 0
Views: 1,106
Posted By mboth
1000 barcodes

Has anybody got any experience in pooling 1000 barcodes?
I am planning a nested multiplex PCR, testing several loci in 1000 clonal colonies for indels. Second PCR should introduce barcodes. IDT...
Forum: Academic/Non-Profit Jobs 05-11-2017, 10:04 AM
Replies: 0
Views: 1,179
Posted By mboth
NGS library prep facility in Cambridge UK is looking for a Research Assistant

http://www.jobs.cam.ac.uk/job/13622/

We are seeking a molecular biologist with proven skills in RNA/DNA manipulation and a working knowledge of DNA sequencing technologies.

The applicant will...
Forum: Sample Prep / Library Generation 06-16-2015, 07:59 AM
Replies: 3
Views: 1,308
Posted By mboth
Thank you for your thoughts so far. Of course I...

Thank you for your thoughts so far.
Of course I had a great clean up, and hope that this will resolve the issue.
The data that comes back shows that some samples were victim of over-amplification...
Forum: Sample Prep / Library Generation 06-11-2015, 03:55 AM
Replies: 3
Views: 1,308
Posted By mboth
problems with sample degradation in the lab

I have experienced some problems in the lab recently, and I am interested if anybody has any comment or suggestions.
We have noticed lower than usual yields for library preps. Now some of the data...
Forum: Sample Prep / Library Generation 06-20-2013, 04:46 AM
Replies: 48
Views: 31,891
Posted By mboth
I have not used Nextera at all, but I was...

I have not used Nextera at all, but I was intrigued by the comment that gDNA must be accurately quantified so that the tagmentation procedure works properly.
I have had a problem with accurately...
Forum: Sample Prep / Library Generation 01-21-2013, 02:05 AM
Replies: 12
Views: 8,147
Posted By mboth
Hello there, I would like to revive this thread...

Hello there, I would like to revive this thread because the same thing happened to us now as well. ChIP-Seq data shows no distinguishable peaks, but qPCR of both the sample and the library confirm...
Forum: Sample Prep / Library Generation 07-17-2012, 05:00 AM
Replies: 3
Views: 4,248
Posted By mboth
Somebody posted this direct link to the Illumina...

Somebody posted this direct link to the Illumina customer letter before. I believe you find your answer in here.
https://icom.illumina.com/Download/Summary/5kKzNLtAtUCIhbHYc3NkUw
Forum: RNA Sequencing 05-03-2012, 07:23 AM
Replies: 1
Views: 1,900
Posted By mboth
I have tried the MirVana Kit from...

I have tried the MirVana Kit from Ambion/LifeTech. That works well and gives you everything below 200nt.
Forum: Sample Prep / Library Generation 03-06-2012, 03:51 AM
Replies: 3
Views: 7,502
Posted By mboth
I have just run a test on our AMPure XP beads,...

I have just run a test on our AMPure XP beads, between new beads with ones that were 4 months over their expiry date. I compared different titrations on a ladder, and saw no difference whatsoever!
Forum: Sample Prep / Library Generation 11-10-2011, 05:42 AM
Replies: 2
Views: 1,990
Posted By mboth
Thank you for the answer ijivanjee! I think it is...

Thank you for the answer ijivanjee! I think it is what I was looking for...
Forum: Sample Prep / Library Generation 11-03-2011, 09:24 AM
Replies: 2
Views: 1,990
Posted By mboth
random primers with three bases?

Does anybody know whether/where I can buy random primers with only 3 bases (A,C,T) for bisulfite-converted cDNA production?
Forum: Sample Prep / Library Generation 10-13-2011, 02:59 AM
Replies: 5
Views: 12,282
Posted By mboth
Thanks everybody! I guess the same applies to...

Thanks everybody!
I guess the same applies to the RNA-seq protocol, where having a large variety of molecules is even more important than having them all the same size.
Has anybody ever seen a...
Forum: Sample Prep / Library Generation 10-06-2011, 08:49 AM
Replies: 1
Views: 2,036
Posted By mboth
On second thoughts, it seems that the ends of RNA...

On second thoughts, it seems that the ends of RNA are being modified when submitted to bisulfite/alkali treatment, forming a 2′,3′ cyclic phosphate (J. Am. Chem. Soc., 1999, 121 (23), pp 53645372),...
Forum: Sample Prep / Library Generation 10-05-2011, 04:13 AM
Replies: 1
Views: 2,036
Posted By mboth
Any advice on RNA bisulfite sequencing?

Hi everyone,
We are planning to treat total RNA (rRNA removed) with bisulfite and convert the nonmethylated cytosines to uracil. This is a harsh treatment and will brake up mRNA into very small...
Forum: Sample Prep / Library Generation 10-04-2011, 02:27 AM
Replies: 5
Views: 12,282
Posted By mboth
Thanks arolfe, that does make sense.

Thanks arolfe, that does make sense.
Forum: Sample Prep / Library Generation 09-29-2011, 09:05 AM
Replies: 5
Views: 12,282
Posted By mboth
advice on the size selection step in ChIP-seq protocol

Hi everyone,
This may be a really silly question, but why do we need to size-select the ChIP sample before PCR amplification? To me it makes more sense to keep as many molecules of the sample and...
Forum: Illumina/Solexa 09-14-2011, 02:23 AM
Replies: 5
Views: 2,897
Posted By mboth
I have done a few TruSeq small RNA library preps....

I have done a few TruSeq small RNA library preps. You are adding ca 120bp of adapters/primers to your fragments, so the tRNA peak is at 180 - 190bp, not at 270. I suspect that 270bp represents rRNA....
Forum: Core Facilities 12-08-2010, 02:24 AM
Replies: 16
Views: 9,627
Posted By mboth
I agree with the previous answers, and would...

I agree with the previous answers, and would probably add the Covaris as well.
Forum: Sample Prep / Library Generation 11-14-2010, 02:47 PM
Replies: 5
Views: 5,878
Posted By mboth
Hello Gord, Thanks for this insight. Would it...

Hello Gord,
Thanks for this insight. Would it be possible to pick your brains regarding the protocols for this endeavour? I am doubtful about several steps, eg shearing, the adapter ligation,...
Forum: Sample Prep / Library Generation 11-11-2010, 06:57 AM
Replies: 5
Views: 5,878
Posted By mboth
Thank you for the comments krobinson and...

Thank you for the comments krobinson and mrawlins! I think we are in a (long) queue for the PacBio machine, and the current strategy is RT at high temperatures. Fingers crossed...
Forum: Sample Prep / Library Generation 11-09-2010, 06:23 AM
Replies: 5
Views: 5,878
Posted By mboth
tRNA sequencing?

Hi,
I was wondering whether anybody has ever wanted to sequence tRNAs? I would be grateful for any information.
I am under the impression cDNA synthesis would be impaired by the modifications and...
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